Whether looking to perform gene overexpression studies or validate gene knockdown/knockout results from your RNAi/CRISPR experiments, LentiORFs are your ideal shortcut to protein expression and tool for gene analysis. Researchers need to validate “hits” from a screen to confirm to eliminate false positives. This means confirming whether the observed phenotype is due the target gene of interest. Re-expressing the gene of interest through ORFs is the best and most accepted way to validate a gene target.

Sigma now offers the complete functional genomics workflow from gene knockdown (siRNA or shRNA), to knockout (CRISPR or ZFN), to gene validation (ORFs) for all your research needs.

What is an ORF?

An open reading frame (ORF) is a continuous segment of DNA beginning with an initiation codon, methionine ATG, and ending with one of the three termination codons, TAA, TAG or TGA, that is coded into a polypeptide chain or a protein. An ORF contains the coding sequence of a gene (CDS) and lacks both the 5’ and 3’ UTRs. These ORFs are inserted into expression vectors (containing an artificial stop codon) and their expression may be modulated (overexpressed) to understand the corresponding gene/protein function. ORFs also play an important role in RNAi and CRISPR rescue experiments, where gene expression is restored (gain-of-function) for gene validation.

MISSION TRC3 Human LentiORF Collection

Sigma offers over 50,000 pre-cloned human ORF constructs with over 14,000 human genes that may be used for overexpression, or rescue and validation experiments. It contains the exclusive puromycin library along with the expanded Human ORFeome v8.1 library (Entry) and CCSB-Broad Lentiviral Expression Library (Blasticidin). See below for library background.

ORF Background: Mission TRC3 (Entry and Blasticidin Libraries)

Original library includes 16,172 clonal ORFs that map to 13,833 human genes, out of which, 14,524 clones (90%) for 12,940 genes (94%) are fully sequenced

Used in applications where rapid ORF shuttling into any Gateway-compatible expression vector is required.

“Open” ORFs omitting the native stop codon

The Mission TRC3 Entry Library includes expanded gene and clone content from the hORFeome v8.1 library, boosting coverage up to 14,000+ human genes total (16,000+ clones).

Figure 1. The Gateway™cloning system(Invitrogen)allows the transfer of DNA fragments between plasmids containing the “Gateway att” flanking sites. A “Gateway entry clone” is prepared by inserting the DNA fragment/gene of interest in between ‘att’ flanking repeats in a plasmid.

The gene of interest from the Gateway Entry clone can be transferred to any Gateway Destination vector (that contains ‘att’ sticky ends, promoter, epitope tag, selection marker) to produce an ‘Expression clone’ for further applications.

Created by arrayed gateway transfer of the entire hORFeome V8.1 collection into the lentiviral vector pLX304-Blast-V5 vector

Vector has a selective marker gene for Blasticidin (Blast) resistance and encodes a C-terminal V5-epitope tag that can be recognized by specific antibodies and useful for Western blotting, immunofluoresence and immunoprecipation experiments

High-throughput, large-scale screening capabilities and expression of thousands of ORFs

Save time: Skip PCR, cloning, and DNA sequence verification steps

The Mission TRC3 Advantage

Although cDNA synthesis is commonly performed in research labs, it can be very costly in terms of time and money. Creation of your own clones is error prone and can take several steps over many days, and require sequence verification. Additionally, mutations may be introduced, and experiments may have to be repeated to get the desired transcript.

Skip sequence verification, PCR, ligation and transformation, and other time-consuming steps (Figure 2 below). Pre-cloned Mission TRC3 LentiORFs take out the uncertainty of de novo cloning and allow you to do your research experiments faster. Our online ordering tool allows you to search for ORF clones, and select your best match type for your gene(s) of interest. Sigma’s fully sequenced LentiORF clones are your ideal shortcut to protein expression, allowing you to focus on your research instead of creating the tools needed to do your research.

Three different Gateway™ adapted LentiORF Vectors

MISSION TRC3 Human LentiORF collection includes a comprehensive collection of fully sequenced ORFs that are packaged in 3 different Gateway-adapted vectors: Entry vector, Blasticidin vector, and Puromycin vector. Custom vector options are also available (see below). All clones are available in glycerol stock, DNA format or viral particle format (excluding the entry library).

The Entry-Vector LentiORF Library

Collection of 16,000+ human ORFs

Created by transferring fully sequenced ORFs from Mammalian Gene Collection (MGC) cDNAs into a recombinational entry vector pDONR223 by adapting the Gateway cloning system

Fusion-ready expression

Lack the native stop codon

Can be easily and efficiently transferred to any Gateway-adapted expression vector containing different promoters, selection markers, fluorophores, tet-inducibility, etc.

Not available in virus format

Blasticidin vector LentiORF Library

Collection of 16,000+ human ORFs

Pre-cloned and ready-to-use

Prepared by inserting the ORF of interest in TRC3 LentiORF-Blasticidin-V5 vector pLX304 using Gateway recombination

For overexpression of the ORF construct in almost any mammalian cell type

Expression-ready vector that has a CMV promoter, which initiates strong expression of the ORF insert

Blasticidin resistant gene acts as a selective marker

Puromycin vector LentiORF Library

Collection of 17,000+ human ORFs

Pre-cloned and ready-to-use

Prepared by inserting the ORF of interest in TRC3 LentiORF-Puromycin-V5 vector pLX317 using Gateway recombination

For overexpression of the ORF construct in almost any mammalian cell type

Expression-ready vector has a EF1-alpha promoter instead of CMV promoter

Preferred for some cell types like primary cells where the CMV promoter gets turned off or methylated

Puromycin resistant gene acts as a selective marker

Exclusive to Sigma

Pooled screening applications: Contains a 24 nucleotide barcode sequence located downstream of the V5 tag and translation stop. Each clone’s unique barcode sequence can be found in the library inventory file for easy identification upon deconvolution.

Our LentiORF expression vector systems allow for additional peptide sequences or tags to modify properties of the native protein. Affinity tags can be used to purify the protein further for further structural and characterization studies. Epitope tags (such as V5 found in the blasticidin and puromycin vectors) allow for simple detection or purification via antibodies. The V5 epitope tag is also useful for western blotting, immunofluoresence and immunoprecipation experiments. The addition of reporters and fluorescent tags also allows for gene expression monitoring and cellular localization.

Contains the LentiORF clone packaged inside a lentivirus that can be directly used for transduction into desired cell lines to carry out gene expression studies

High titer and high volume options available (10^5-10^9, 0.1ml-10 ml)

Figure 3. Upon viral transduction, the LentiORF expression plasmid DNA integrates into the nucleus of the host cell. The cellular machinery recognizes the promoter and transcribes the protein. The resulting gene product and encoded protein can then be used for purification, localization for characterization of protein function, or to determine the altered phenotype as a result of gene overexpression.

How are the ORFs classified?

Depending on how close the sequences of the ORFs match or align with NCBI Refseq transcript, they are classified as perfect match, near-perfect, lower, and other.

Perfect Match: Perfect alignment to the full open reading frame (100%).

Near-Perfect Match: The ORFs in this category align nearly perfectly to the full open reading frame of at least one, and usually exactly one, RefSeq transcript. 70% of fully-sequenced clones have a 99% match over 99% of the length to RefSeq.

Lower Match: About half of the ORFs in this category have one or more clear but partial matches to RefSeq (at least a 90% match over at least 50% of the length of the clone).

Gene Rescue/Validation

RNAi Rescue

RNA interference is used for the functional assessment of human genes that mediate cellular phenotypes by conducting loss-of-function gene studies. In addition to gene specific silencing through perfect complementarity to the target transcript, RNAi can cause phenotypic changes by the regulation of unintended transcripts through seed-region complementarity that can lead to false interpretation of target gene function.

Therefore, it becomes necessary to validate target specificity of siRNA or shRNA phenotypes via RNAi rescue experiments. One such method of phenotypic rescue involves the use of exogenously expressed shRNA/siRNA-insensitive transcript.

These transcripts can be obtained from the TRC3 Human LentiORF collection that contains ORFs lacking the 3’UTR and 5’UTR. This makes the ORFs resistant to siRNA or shRNAs that target the 3’UTR of the gene transcript. The siRNA/shRNA will silence only the endogenous expression of the gene of interest.

Therefore, these ORFs can be used to express exogenously in order to permit phenotypic rescue as a means of validating gene function. If an exogenous expression of the ORF for the gene of interest results in phenotype rescue, it is a confirmation that the phenotype is due to target-specific effects of the shRNA/siRNA. If the phenotypic effect is still observed, it indicates that the shRNA/siRNA effect is non-specific or off-target. The ORF constructs can be transfected as plasmids or transduced as lentiviral particles in difficult-to-transfect cells.

Additionally, the level of expression of the ORF can be regulated by controlling the ratio of functional viral particles to cells.

Another method of selective deletion/inactivation of genes is by the CRISPR/Cas9 system (Clustered regularly interspaced short palindromic repeats/CRISPR associated 9 system). It is a two-component system involving the Cas9 and guide RNA (gRNA). Cas9 nuclease produces double strand breaks in the genomic target site. Binding specificity is based on the gRNA and a three nucleotide NGG sequence called the protospacer adjacent motif (PAM) sequence. (Marraffini & Sontheimer, 2010)

Benefits of CRISPR/Cas9 system

Enables the inactivation of genes in somatic cells. Homologous recombination in the germline is not required.

Works more efficiently when compared to RNAi with complete inactivation of the target gene/knockout of the target gene. RNAi/knockdown may lead to incomplete gene silencing resulting in residual protein that makes the interpretation of the resulting phenotype difficult. (Incontro S, 2014)

The requirement for an NGG sequence makes target design simple and straightforward in genomic regions where off-targeting is not an issue

“The relatively acute effect of CRISPR/Cas9 further mitigates the potential for compensation. In addition, it is essential to perform rescue experiments that definitively rule out off-target effects as the cause of the observed phenotype. For the rescue, it is important to ensure that the rescue construct will not itself be targeted by CRISPR. This can be achieved either by using a cDNA with silent mutations introduced into the region recognized by the gRNA (and/or the PAM) or by using a cDNA from a different species if there is sufficient divergence in the sequence. In this study, we performed the rescue experiment using another, simpler approach. Indeed, we searched for a specific gRNA sequence encompassing an intron-exon junction such that the cleavage site (position 3) would be in the exon, but most of the gRNA sequence would lie within the intron. By using this very simple strategy, it is now possible to perform rescue experiments using unmodified cDNA, which greatly simplifies and accelerates the validation of the phenotype."

Gene Overexpression Studies

While RNA interference technology allows scientists to “turn off” or silence certain genes, ORFs can be used to “turn on” or “overexpress” particular genes. Flipping the switches on genes one at a time can help reveal the functions of individual genes, such as those that play a role in cancer. The lentiviral expression system has been used in several studies where gene screening, expression analysis and genetic rescue are important. Over-expression of genes and proteins is widely used in functional genomics, proteomics and system biology studies.